Polymer modified porous substrate for solid phase extraction

ABSTRACT

A polymer modified porous substrate for solid phase extraction or chromatography, comprising a porous substrate and a polymeric monolith formed thereon, wherein the polymeric monolith comprises C 5-10  monocyclic or bicyclic aryl or heteroaryl, optionally substituted with -L-Q p -R q ; where q is 0-3, p is 0-5, Q is —NRC(O)—, —C(O)NR—, —OC(O)NR—, —OC(O)R—, —NRC(O)O—, —NRC(O)NR—, —NCO, —CHOHCHOH—, CH 2 OCHCH 2 O—, —(CH 2 CH 2 O) s —, —(CH 2 CH 2 CH 2 O) s —, —C(O)—, —C(O)O—, —CH 2 C(O)CH 2 —, —S—, —SS—, —CHOH—, —O—, —SO—, —SO 2 —, —SO 3 —, —OSO 3 , —SO 2 NR—, —NR q —, and —NR q   + —, —CN, —NC, —CHOCH—, —NHC(NH)NH—, —NO 2 , —NO, —OPO 3 —, —OH; and R is hydrogen, C 5-10  monocyclic or bicyclic aryl or heteroaryl, C 1-12  branched, unbranched, or cyclic hydrocarbyl; L is a bond or a C 1-12  branched, unbranched, or cyclic hydrocarbyl. Methods of preparing and using the polymer modified porous substrates are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of the U.S. patentapplication Ser. No. 11/120,003, filed on May 2, 2005 now U.S. Pat. No.7,311,825 entitled POLAR FUNCTIONALIZED POLYMER MODIFIED POROUSSUBSTRATE FOR SOLID PHASE EXTRACTION.

FIELD OF THE INVENTION

This invention relates generally to devices and methods for analyticaland preparatory separations and the like.

BACKGROUND OF THE INVENTION

The use of polymeric sorbents is known in the area of analytical andpreparative separations. The porous structure can be controlled bychoice of monomers, amount of crosslinking copolymer, polymerizationtemperature, and the amount and type of porogenic solvent. Theseparameters and their affect on monolith pore structure are reviewed indetail in Okay, O. Prog. Polym. Sci. (2000) 25:711-779.

One commonly used polymeric monolith is formed from the free radicalpolymerization of styrene and divinylbenzene. Many examples ofstyrene/divinylbenzene polymers are known in the art, and have beenapplied in the areas of solid phase extraction and chromatography. Forexample, Premstaller, A. (2000) Anal. Chem. 72:4386 describes theperformance of a monolithic chromatography bed comprising poly(styrenedivinylbenzene) and compared it with a column packed withmicropellicular poly(styrene divinylbenzene) polymer beads. A monolithicpoly(styrene-divinylbenzene) was formed in capillary tubing and used toseparate double stranded nucleic acids in preparation for electrosprayionization mass spectrometry. The monolithic column exhibited animprovement in column performance relative to a column packed withpolymer beads.

Merhar, M. et al. (2002) Materiali in Tehnologije 36:163 describe arepresentative polymer monolithic disk and its use to separate a mixtureof macromolecules (proteins).

US 2003/0229191 to Kallury describes a polymeric sorbent comprising apolymeric backbone adapted to facilitate one or more interactionsselected from the groups consisting of a dipolar interaction and ahydrophobic interaction and an amide functionality associated withpolymeric backbone and adapted to undergo one or more interactionsselected from the group consisting of proton accepting, proton donatingand dipolar interactions, and exhibiting a strong capacity for retentionof polar molecules. The sorbent can be associated with supportsincluding disks, membranes, and syringe barrel cartridges for samplepretreatment.

U.S. Pat. No. 5,616,407 to Fritz describes a functionalized macroporouspoly(styrene divinylbenzene) particle comprising ionic functional groupsfor adsorbing analytes. Similarly, EP 0758261B1 and U.S. Pat. No.5,618,438 to Fritz describe the use of the aforementioned macroporouspoly(styrene divinylbenzene) particle in a solid phase extraction mediumcomprising a fibrous matrix and sorptive particles enmeshed in thematrix in a weight ratio of sorptive particles to fibrous matrix of 40:1to 1:4. EP 498557A1 describes a method for preparing a solid phaseextraction medium comprising a PTFE fibril matrix and sorptive particlesenmeshed in said matrix and a method for isolating an analyte. The solidphase extraction medium is prepared by blending the particles with aPTFE emulsion and subjected to mixing to cause the fibrillation of thePTFE particles, and calendared to form a calendered sheet. The particlesare described as being separate from each other and isolated in a PTFEfibril cage that restrains the particle.

U.S. Pat. No. 5,738,790 to Hagen describes a solid phase extractionmedium comprising a porous nonwoven fibrous matrix comprising PTFE andblown microfibers and sorptive or reactive hydrophobic siliceousmolecular sieve particles enmeshed in said matrix in a weight ration of40:1 to 1:40.

Variations on the monomers that can be used in the preparation ofpolymeric sorbents have also been investigated. For example, EP 1159995and U.S. Pat. No. 6,759,442 to Takahashi describe a packing material forsolid phase extraction of hydrophobic and ionic substances, reportedlyhaving hydrophobicity and an ion exchange group. The packing material isdescribed as a particle obtained by copolymerizing a hydrophobic monomer(A) and a hydrophilic monomer (B) and introducing thereinto an ionexchange group, in which the ion exchange group is introduced allegedlywithout impairing the hydrophobic site.

U.S. Pat. No. 6,322,695 to Lee describes a porous resin comprisingcrosslinked polymer particles penetrated by channels for solid phaseextraction. The polymeric particles are said to feature a hydrophobiccomponent, at least one hydrophilic component and at least one ionexchange functional group. In certain embodiments, the hydrophobicmonomer is divinylbenzene, the hydrophilic monomer isN-vinylpyrrolidone, and the copolymer is a sulfonatedpoly(divinylbenzene-co-N-vinylpyrrolidone).

U.S. Pat. No. 6,749,749 to Xie describes the preparation of permeablepolymeric monolithic materials in column casings wherein the applicationof pressure allegedly avoids wall effects and swelling. In particularembodiments, filler materials such as polymer rods or silica beads areused as a framework for the polymer and allegedly provide greatermechanical strength.

Tsuda et al. in U.S. Pat. No. 6,723,157 and (2003) Analytical Sciences20, 1061 describe the preparation of a type of fiber adsorbent havingattached silica microparticles for adsorbing gaseous toluene at lowconcentrations. The silica microparticles are reportedly prepared bypolymerizing silica oligomers under alkaline conditions and fixing themonto glass fibers, which were woven into a glass fiber. The surface ofthe silica microparticles was chemically modified by bonding C₁₈ phases.

EP 0432438 describes molded adsorbents comprising a mixture of adsorbentparticles, fine plastic particles and reinforcing fibers. The adsorbentis activated carbon, silica, alumina, or zeolites. U.S. Pat. No.4,512,897 to Crowder describes a molecular separation column foreffecting the differential distribution between two phases, the columncontaining a substantially homogenous solid stationary phase whichcomprises a porous matrix of fiber having particulate immobilizedtherein.

However, the polymeric sorbents described in the art are provided asparticles which must be incorporated into a sorbent bed or enmeshed infiber networks. The resultant articles are not convenient andinexpensive to manufacture, nor do they provide ease of use in solidphase extraction applications. The solid phase adsorptioncharacteristics are limited, and the sorbents do not provide goodretention of both polar and nonpolar analytes. In addition, the solidphase extraction media do not provide recovery of analytes in smalleluant volumes with superior flow rates, allowing fast and efficient useof time, labor and solvents in analytical applications.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the invention to address theaforementioned needs in the art by providing a polymer modified poroussubstrate for solid phase extraction or chromatography.

It is an additional object of the invention to provide improved methodsfor solid phase extraction.

It is an additional object of the invention to provide improved methodsfor preparing solid phase extraction media.

Accordingly, a polymer modified porous substrate for solid phaseextraction or chromatography is provided, comprising a porous substrateand a polymeric monolith formed thereon, wherein the polymeric monolithhas the formula

wherein A is selected from C₅₋₁₀ monocyclic or bicyclic aryl orheteroaryl, optionally substituted with -L-Q_(p)-R_(q); q is 0-3; p is0-5, preferably 0-2; Q is —NRC(O)— (amide), —C(O)NR— (carbamyl),—OC(O)NR— (carbamate), —OC(O)R (alkyloxy), —NRC(O)O— (urethane),—NRC(O)NR— (carbamide or urea), —NCO (isocyanate), —CHOHCHOH— (diol),CH₂OCHCH₂O— (glycidoxy), —(CH₂CH₂O)_(s)— (ethoxy), —(CH₂CH₂CH₂O)_(s)—(propoxy), —C(O)— (carbonyl), —C(O)O— (carboxy), —CH₂C(O)CH₂—(acetonyl), —S— (thio), —SS— (dithio), —CHOH—, —O— (ether), —SO—(sulfinyl), —SO₂— (sulfonyl), —SO₃— (sulfonic acid), —OSO₃ (sulfate),—SO₂NR— (sulfonamide), —N_(q)—, (amines), and —NR_(q) ⁺—, where R is notH (quaternary amines), —CN (nitrile), —NC (isonitrile), —CHOCH— (epoxy),—NHC(NH)NH— (guanidino), —NO₂ (nitro), —NO (nitroso), —OPO₃—(phosphate),—OH (hydroxy), and s is 1-12; and R is hydrogen, C₅₋₁₀ monocyclic orbicyclic aryl or heteroaryl, C₁₋₂ branched, unbranched, or cyclichydrocarbyl;

P is

L is a bond or a C₁₋₁₂ branched, unbranched, or cyclic hydrocarbyl; andwherein the order of [—CH₂—CR-L-A-P] and [—CH₂—CR-L-A] is random, blockor a combination thereof. Preferably, n/m is from 0 to 1000 or m/n isfrom 0 to 1000; The porous substrate can be in the form of a monolith,agglomerated particles, or woven or nonwoven fibers. A preferred poroussubstrate is a glass fiber monolith. The polymeric monolith canoptionally be functionalized with a polar functionality after thepolymeric monolith is formed on the porous substrate to provide desiredchemical surface characteristics such as wettability, or a desiredanalyte retention.

Methods for preparing the polymer modified porous substrate generallycomprise: (a) contacting a porous substrate with a solution comprisingone or more hydrophobic monomers, a porogenic solvent and apolymerization initiator; and (b) heating the porous substrate andretained solution in the absence of oxygen to polymerize the one or moremonomers onto the porous substrate. In certain embodiments, thehydrophobic monomers are crosslinkable monomers, uncrosslinkablemonomers, or combinations thereof. It is preferred that the hydrophobicmonomer has the formulaCH₂═CR-L-A-L_(r)[—CR═CH₂]_(r)where L is a bond or a C₁₋₁₂ branched, unbranched, or cyclichydrocarbyl; A is a C₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl,optionally substituted with C₁₋₁₂ branched or unbranched hydrocarbyl,halo, and r is 0 or 1. Preferably, the uncrosslinkable monomer isselected from vinyl or allyl substituted C₅₋₁₀ monocyclic or bicyclicaryl or heteroaryl, optionally substituted with C₁₋₁₂ branched orunbranched hydrocarbyl, halo, or combinations thereof. Preferably, thecrosslinkable monomer is selected from divinyl- or diallyl-substitutedC₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl moieties, optionallysubstituted with C₁₋₁₂ branched or unbranched hydrocarbyl, halo, orcombinations thereof.

The method can further comprising treating the polymer modified poroussubstrate to introduce a polar functionality selected from —NRC(O)—,—C(O)NR—, —OC(O)NR—, —OC(O)R, —NRC(O)O—, —NRC(O)NR—, —NCO, —CHOHCHOH—,CH₂OCHCH₂O—, —(CH₂CH₂O)_(s)—, —(CH₂CH₂CH₂O)_(s)—, —C(O)—, —C(O)O—,—CH₂C(O)CH₂—, —S—, —SS—, —CHOH—, —O—, —SO—, —SO₂—, —SO₃—, —OSO₃,—SO₂NH—, —SO₂NR—, —NR_(q)—, and —NR_(q) ⁺—, —CN, —NC, —CHOCH—,—NHC(NH)NH—, —NO₂, —NO, —OPO₃—, —OH; wherein s is 1-12; q is 0-3; and Ris hydrogen, C₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl, C₁₋₁₂branched, unbranched, or cyclic hydrocarbyl. The porous substrate can bein the form of a monolith, agglomerated particles, or woven or nonwovenfibers.

In an additional embodiment, a method is provided for preparing an amidefunctionalized polymer modified porous substrate comprising: (a)contacting a porous substrate with a solution comprising one or morehydrophobic monomers, a porogenic solvent and a polymerizationinitiator; (b) heating the porous substrate and retained solution in theabsence of oxygen to polymerize the one or more monomers onto the poroussubstrate to form the polymer modified porous substrate, (c) nitratingthe polymer modified porous substrate to form the nitrated polymermodified porous substrate; (d) reducing the nitrated polymer modifiedporous substrate to form the aminated polymer modified porous substrate;and (e) acylating the aminated polymer modified porous substrate with anacid, an acid chloride or an acid anhydride to form the amidated polymermodified porous substrate. In one embodiment, the method can furthercomprise sulfonating the amidated polymer modified porous substrate. Inan additional embodiment, the method can further comprise treating theaminated or amidated polymer modified porous substrate to form aquaternary amine functionalized polymer modified porous substrate.

In an additional aspect, methods are provided for isolating an analyte,comprising (a) conditioning a polymer modified porous substrate with anorganic solvent and optionally an aqueous solvent, or mixtures thereof;(b) adsorbing analytes present in a sample to be analyzed to theconditioned polymer modified porous substrate; and (c) eluting theadsorbed analytes from the polymer modified porous substrate with anorganic solvent, an aqueous solvent, or mixtures thereof.

In another aspect, a method is provided for performing a chromatographicseparation of analytes, comprising a) providing a polymer modifiedporous substrate disposed in a chromatography apparatus; b) conditioningsaid polymer modified porous substrate with an organic solvent, aqueoussolution, or mixtures thereof; c) contacting said polymer modifiedporous substrate with a solution comprising one or more analytes; d)passing a mobile phase comprising an organic solvent, a aqueoussolution, or mixtures thereof, through said polymer modified poroussubstrate; and e) eluting one or more analytes from the polymer modifiedporous substrate.

In an additional embodiment, devices for performing solid phaseextraction or chromatography are provided, comprising a polymer modifiedporous substrate associated with a support. Preferably, the support is asyringe barrel cartridge, a chromatography column, a microfluidicsplatform, one or more additional membranes, a pipette tip or amultiwelled plate

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic for the preparation of a polymer modifiedporous substrate.

FIG. 2 illustrates a schematic for the preparation of an amidefunctionalized polymer modified porous substrate.

FIG. 3 illustrates the relative retention of analytes of varyingpolarity by various polymer modified porous substrates.

FIG. 4 illustrates the elution of analytes from one polymer modifiedporous substrate.

FIG. 5 illustrates the elution of analytes from a second polymermodified porous substrate.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions and Overview

Before the present invention is described in detail, it is to beunderstood that unless otherwise indicated this invention is not limitedto specific analytes, solvents, chemistries, or the like, as such mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only and is notintended to limit the scope of the present invention.

It must be noted that as used herein and in the claims, the singularforms “a,” “and” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a solvent”includes two or more solvents; reference to “an analyte” includes two ormore analytes, and so forth.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

As used herein, the term “adsorb” and grammatical derivatives thereof,means a surface phenomena wherein an analyte becomes reversiblyassociated with the surface of a polymeric sorbent by physicallyinteracting with the surface molecules. The association can be, forexample, via any non-covalent mechanism such as van der Waal's forces,dipole-dipole interactions, dipole-induced dipole or dispersive forces,via hydrophobic interactions or hydrogen donor or acceptor interactions,or including electrostatic or ion exchange interactions.

As used herein, the term “analyte” means any molecule to becharacterized, identified or quantitated in a sample of biological,organic, synthetic, natural or inorganic origin. For example, acandidate therapeutic compound or metabolite thereof can be an analyte,and can be present in, for example, a blood plasma sample, saliva,urine, drinking water, mixture of synthetic or natural products, orenvironmental sample. An analyte can exhibit any polarity, from nonpolarto polar.

As used herein, the term “monolith” means a continuous structure,including for example, honeycomb structures, foams, and fibers,including fibers woven into fabrics or made into non-woven mats or thinpaper-like sheets. Foams generally exhibit a sponge-like structure.

As used herein, the term “substrate” refers to a porous substantiallyinsoluble material that is stable to washing or reaction conditions,such as those encountered during sample preparation and/or syntheticprocedures, including solvent extraction steps and in situ reactionsteps.

The terms “polar,” “nonpolar” and “polarity” refer generally to thepartition coefficient P of a compound of interest, which is the ratio ofthe equilibrium concentrations of the compound in an organic phase(e.g., octanol) relative to an aqueous phase (e.g., water). As usedherein, a polar compound is generally characterized as having a log Pvalue less than 2.0, while a nonpolar compound is generallycharacterized as having a log P value greater than about 2.0.

The term “polar functionality” generally refers to the followingchemical groups: —NRC(O)— (amide), —C(O)NR— (carbamyl), —OC(O)NR—(carbamate), —OC(O)R— (alkyloxy), —NRC(O)O— (urethane), —NRC(O)NR—(carbamide or urea), —NCO (isocyanate), —CHOHCHOH— (diol), CH₂OCHCH₂O—(glycidoxy), —(CH₂CH₂O)_(s)— (ethoxy), —(CH₂CH₂CH₂O)_(s)— (propoxy),—C(O)— (carbonyl), —C(O)O— (carboxy), —CH₂C(O)CH₂— (acetonyl), —S—(thio), —SS— (dithio), —CHOH—, —O— (ether), —SO— (sulfinyl), —SO₂—(sulfonyl), —SO₃— (sulfonic acid), —OSO₃ (sulfate), —SO₂NMe—(sulfonamide), —NR_(q)—, (amines), and —NR_(q) ⁺—, where R is not H(quaternary amines), —CN (nitrile), —NC (isonitrile), —CHOCH— (epoxy),—NHC(NH)NH— (guanidino), —NO₂ (nitro), —NO (nitroso), —OPO₃—(phosphate), —OH (hydroxy), where s is greater than 1, or morepreferably 1-12; and R is hydrogen, C₅₋₁₀ monocyclic or bicyclic aryl orheteroaryl, C₁₋₁₂ branched, unbranched, or cyclic hydrocarbyl.

The term “hydrocarbyl” is used in its conventional sense to refer to ahydrocarbon group containing carbon and hydrogen, and may be straight orbranched chain aliphatic, alicyclic or aromatic, or may contain acombination of aliphatic, alicyclic and/or aromatic moieties. Aliphaticand alicyclic hydrocarbyl may be saturated or they may contain one ormore unsaturated bonds, typically double bonds. When a hydrocarbylresidue having a specific number of carbons is named, all geometricisomers having that number of carbons are intended to be encompassed;thus, for example, “butyl” is meant to include n-butyl, sec-butyl,isobutyl and t-butyl; “propyl” includes n-propyl and isopropyl.

The present inventors have surprisingly discovered that adsorbentpolymers can be formed directly on porous substrates to form a polymermodified porous substrate which provides a superior medium for solidphase extraction or chromatography. The polymer modified poroussubstrates are simple to use and manufacture, and provide a significantadvance in the art of solid phase extraction of analytes. When thepolymer is functionalized with one or more polar moieties, superiorretention of polar analytes and methods for recovering analytes ofparticular polarities are provided. The polymer monolith formed on theporous substrate provides superior results in comparison with prior artdevices described as comprising entrapped polymeric particles. Thepolymer modified porous substrate also provides improved methods ofisolating analytes and performing solid phase extraction.

The components and uses of the polymer modified porous substrates aredescribed in greater detail below.

II. Polymers

The polymeric monolith which is formed on the polymer modified poroussubstrate comprises hydrophobic moieties to adsorb nonpolar compounds,and in certain embodiments, comprises one or more polar functionalitiesto provide enhanced retention of polar compounds. The polymeric monolithhas the formula

wherein A is selected from C₅₋₁₀ monocyclic or bicyclic aryl orheteroaryl; L is a bond or a C₁₋₁₂ branched, unbranched, or cyclichydrocarbyl;P is

and wherein the order of [—CH₂—CR-L-A-P] and [—CH₂—CR-L-A] is random,block or a combination thereof. The moiety P provides a crosslinkedpolymer bonded with two different polymeric backbones. The polymericmonolith is formed from the polymerization of one or more hydrophobicmonomers, which can be crosslinkable monomers, uncrosslinkable monomers,or combinations thereof. When only crosslinkable monomers are used toprepare the polymer, n is any positive integer and m is zero. Whenuncrosslinkable monomers are included in the polymerization reaction,the ratio m/n can be from 0 to 10000 or more as desired. When onlyuncrosslinkable monomers are used to prepare the polymer, m is anypositive integer and n is zero. When crosslinkable monomers are includedin the polymerization reaction, n/m can be from 0 to 10000 or more.Preferably, when both crosslinkable monomers and uncrosslinkablemonomers are present, they can be used in any molar ratio to achieve adesired amount of crosslinked polymer, for example, a molar ratio of1:1000 to 1000:1, or more preferably 1:100 to 100:1, respectively. In apreferred embodiment, the ratio of crosslinkable monomer touncrosslinkable monomer is from 1:10 to 10:1, for example as disclosedin Example 8.

Enhanced retention of polar compounds can be effected by using apolymeric monolith having capabilities of hydrophilic interactions(e.g., proton accepting, proton donating, dipolar, electrostaticattraction, ion exchange, etc.) as well as hydrophobic interactions. Toprovide enhanced adsorption of polar compounds, the polymer can bemodified with a polar functionality to provide hydrophilic in additionto a hydrophobic interaction. In addition, enhanced adsorption ofcharged compounds can be effected by a polymer having ion exchangecapabilities. To provide a polymer modified porous substrate having ionexchange capabilities, an ion exchange functionality can be introducedonto the polymer. Accordingly, the aryl or heteroaryl moieties can befunctionalized with a polar functionality after the polymeric monolithis formed on the porous substrate, by optionally substituting the arylor heteroaryl moieties with L-Q_(p)-R_(q); wherein q is 0-3; p is 0-5,or more preferably p is 0-2; Q is —NRC(O)—, —C(O)NR—, —OC(O)NR—,—OC(O)R—, —NRC(O)O—, —NRC(O)NR—, —NCO, —CHOHCHOH—, CH₂OCHCH₂O—,—(CH₂CH₂O)_(s)—, —(CH₂CH₂CH₂O)_(s)—, —C(O)—, —C(O)O—, —CH₂C(O)CH₂—, —S—,—SS—, —CHOH—, —O—, —SO—, —SO₂—, —SO₃—, —OSO₃, —SO₂NMe—, —NR_(q)—, and—NR_(q) ⁺— (where R is not H), —CN, —NC, —CHOCH—, —NHC(NH)NH—, —NO₂,—NO, —OPO₃—, —OH, where s is 1-12; and R is hydrogen, C₅₋₁₀ monocyclicor bicyclic aryl or heteroaryl, C₁₋₁₂ branched, unbranched, or cyclichydrocarbyl. The polar functionalized modified porous substrate thusprovided adsorbs analytes utilizing hydrophobic as well as hydrophilicinteractions, which includes at least one interaction selected fromproton accepting, proton donating, dipolar, electrostatic attraction andion exchange interactions.

The polymer can also be formed from a crosslinkable monomer, such asdivinylbenzene, which can provide additional structural strength to thepolymer. The polymer can comprise, for example, poly(styrenedivinylbenzene), copolymers of styrene or divinylbenzene withfunctionalized aryl or heteroaryl moieties such as styrenes orheterocycles carrying substituents such as halo or alkyl. Thus, arepresentative, but non-limiting, list of polymers that can be utilizedas the polymer for modifying the porous substrate includes, but is notlimited to, polystyrene, poly(styrene divinylbenzene), copolymerscomprising styrene or divinylbenzene and halogenated or alkylatedstyrenes, pyridines, thiophenes, furans, imidazoles, and the like.

III. Porous Substrates

The porous substrates can be any porous solid structure providingsufficient solvent and analyte access and porosity (e.g., a density ofno more than about 1 cc/g). Preferably, the porous substrate alsoexhibits substantial inertness so that the porous substrate does notdissolve or erode under the conditions of polymer formation or analyteisolation. Preferably, the porous substrate is in the form of amonolith, agglomerated particles, or woven or nonwoven fibers,preferably nonwoven fibers including macro and microfibrous webs such asmelt-blown webs, spunbonded or air laid webs and blown fibrous webs,such as described in U.S. Pat. No. 5,328,758 and references citedtherein. The porous substrate can be formed from inorganic materialssuch as metal or metalloid oxides (e.g., silica, alumina, titania,zirconia, vanadia, zeolite, mullite, glass, etc.), metals (e.g.,stainless steel), or organic materials such as carbon fibers, cellulosicmaterials (e.g., nitrocellulose, cellulose acetate), synthetic polymersincluding poly(vinylchloride), polyacrylamide, polyacrylate, polyolefins(e.g., polyethylene, polypropylene, polytetrafluoroethylene (PTFE)),poly(4-methylbutene, polystyrene, polyurethanes, polyacrylonitriles,polymethacrylate, poly(ethylene terephthalate), polysiloxanes, nylon,poly(vinyl butyrate), and the like, or mixtures or composites of any ofthe above. The porous substrate does not itself adsorb a significantamount of analytes, due to the polymerization of monomers upon andthroughout the porous substrate. Preferably, the porous substrate is aglass fiber monolith.

In certain embodiments, the porous substrate is inert. By “inert” ismeant that the porous substrate underlying the polymer modified poroussubstrate is sufficiently stable under conditions of use (e.g., analyteisolation and preparation using solvents, acids, bases, salts orbuffers, etc.) such that the polymer modified porous substrate can beused without contamination of the analyte solution. In particularembodiments, the porous substrate is sufficiently inert to be used toprepare an amide functionalized polymer modified porous substrate, forexample, involving the use of strong acids or base, without losingstructural integrity. Typical inert substrates include polymericsubstrates such as polyolefins (e.g., polyethylene, PTFE),polysiloxanes, polystyrenes, and the like, inorganic substrates formedfrom materials such as silica, zirconia, vanadia and alumina, forexample, in the form of glass or ceramic fibers, and the like, andmetals (e.g., stainless steel mesh). Preferably, the substrate is aglass fiber monolith. There is no particular limitation on the type ofglass fiber that can be used. For example, good results have beenobtained with glass fibers having thickness of between about 0.010 and0.050 inches. However, fibers of other thicknesses can be utilized.

The porous substrates can include ultraporous monolithic substratesformed, for example, as described in commonly owned, co-pending U.S.patent application Ser. No. 11/018,168, filed on Dec. 20, 2004. Furtherinert porous substrates can be prepared by forming silica particleswhich can then be coalesced into a porous structure. One variation isdescribed in U.S. Pat. No. 4,017,528 to Unger, which describes a processfor preparing a hybrid silica wherein an alkyl functionality is coupledonto both the skeleton structure and the surface of the silica. Themethod involves forming a mixture of tetraethoxysilane (TEOS) and anorganotriethoxysilane and hydrolyzing the silanes in the presence of anacid catalyst to form polyalkylethoxysiloxane oligomers that are thengelled into porous hybrid particles in the presence of a base catalyst.

Preferably, the porous substrate is in a form that is suitable forperforming solid phase extraction of analytes. Applications arediscussed further below.

IV. Porogenic Solvents

The polymer formed upon the porous substrate is itself porous. Porosityis provided by a porogenic solvent that exhibits the followingproperties: it dissolves in the monomer mixture, is inactive to thepolymerization reaction and does not dissolve the polymer produced.Suitable porogenic solvents include, but are not limited to, aromatichydrocarbons, such as toluene, xylene, ethylbenzene and diethylbenzene;saturated hydrocarbons, such as hexane, heptane, octane and decane;alcohols, such as isoamyl alcohol, octanol, decanol, dodecanol and2-ethylhexyl alcohol; aliphatic halogenated hydrocarbons, such asdichloromethane, dichloroethane and trichloroethane; aliphatic oraromatic esters, such as ethyl acetate, butyl acetate, dimethylphthalate and diethyl phthalate; and glycerol triesters, such astriacetin, tributyrin and tricaprin. The porogenic solvents can be usedindividually or in combination of two or more thereof. The amount of theporogenic solvent added can be varied from about 10% to 300% by massbased on the total amount of the monomers.

V. Polymerization Initiators

Polymerization initiators include radical polymerization initiators suchas benzoyl peroxide, diisopropyl peroxydicarbonate, t-butylperoxy-2-ethylhexanoate, t-butyl peroxypivalate, t-butylperoxydiisobutyrate, lauroyl peroxide, dimethyl 2,2′-azobisisobutyrate(MAIB), azobisisobutyronitrile (AIBN) and azobiscyclohexanecarbonitrile(CAN).

VI. Polar Functionalization of the Polymer Modified Substrate

In certain preferred embodiments, the aryl or heteroaryl moieties of thepolymeric monolith formed on the porous substrate can be functionalizedwith a polar functionality after the polymeric monolith is formed on theporous substrate, by treating the polymer so as to introduce polarfunctionalities such as -L-Q_(p)-R_(q); wherein q is 0-3; p is 0-5, ormore preferably p is 0-2; Q is —NRC(O)—, —C(O)NR—, —OC(O)NR—, —OC(O)R—,—NRC(O)O—, —NRC(O)NR—, —NCO, —CHOHCHOH—, CH₂OCHCH₂O—, —(CH₂CH₂O)_(s)—,—(CH₂CH₂CH₂O)_(s)—, —C(O)—, —C(O)O—, —CH₂C(O)CH₂—, —S—, —SS—, —CHOH—,—O—, —SO—, —SO₂—, —SO₃—, —OSO₃, —SO₂NR—, —NR_(q)— and —NR_(q) ⁺—, —CN(nitrile), —NC, —CHOCH—, —NHC(NH)NH—, —NO₂, —NO, —OPO₃—, —OH, where s isgreater than 1, or more preferably 1-12; and R is hydrogen, C₅₋₁₀monocyclic or bicyclic aryl or heteroaryl, C₁₋₁₂ branched, unbranched,or cyclic hydrocarbyl. The polar functionalized modified poroussubstrate thus provided adsorbs analytes utilizing hydrophobic as wellas hydrophilic interactions, which includes at least one interactionselected from proton accepting, proton donating, dipolar, electrostaticattraction and ion exchange interactions.

In preferred embodiments, the polar functionality is an amide, amine, ornitro. In another preferred embodiment, the polar functionality is acation exchange functionality (e.g., sulfonic acid). In an additionalpreferred embodiment, the polar functionality is an anion exchangefunctionality (e.g., quaternary amino).

VII. Devices

In an additional aspect, there are provided devices for performing solidphase extraction. For example, the polymer modified porous substrate canbe further associated with a support or supporting format. A list ofrepresentative supports and supporting formats includes, but is notlimited to, syringe barrel cartridges, chromatography columns,additional polymeric or glass fiber membranes, microfluidics platform,pipette tips and multiwelled plates, although disks and other supportscan also be employed. The polymer modified porous substrate can bedisposed on the surface of a supporting format, for example on thesurface of a multiwelled plate, or the polymer modified porous substratecan be embedded in a supporting format, for example between polymeric orglass fiber membranes. Thus, by “association” it is generally meant thata sorbent can be in contact with a support or supporting format.

In another aspect, the polymer modified porous substrate can beassociated with a support to facilitate analyte loading and/or elutionwith solvent. A support or a supporting format can have any one of anumber of configurations or shapes, such as strip, plate, disk, hollowtube, rod, and the like. A support or supporting format can behydrophobic, hydrophilic or capable of being rendered hydrophilic.Representative supports and supporting formats include, withoutlimitation, syringe barrel cartridges, pipette tips, multiwelled plates,microfluidics platforms, integrated sample preparation, injection anddetection device, and the like, and can be disposed on a surface of thesupporting device or embedded within a channel or tube within thedevice.

In a preferred embodiment, the polymer modified porous substrate is usedin solid phase extraction devices such as a solid phase extractionpipette, for example, as described in co-pending U.S. Patent ApplicationSer. No. 60/473,996 filed on May 29, 2003 and PCT Application No.US2004/016904 filed on May 28, 2004 to Hudson, which is directed to thepreparation and use of solid phase extraction devices comprising afunctionalized monolithic sorbent comprising a glass fiber matrixembedded with a bonded phase comprising a metal oxide or metalloid oxidehaving reactive metal oxides capable of reacting with silanes. The solidphase extraction pipette typically comprises a hollow tube having onebroader opening and one narrower opening that functions as a pipettetip. One method to prepare the solid phase extraction pipette generallycomprises placing the polymer modified porous substrate in the smalleropening of the pipette by the following steps: inserting the polymermodified porous substrate into the larger opening of the pipette;applying reduced pressure to the smaller opening of the pipette toinsert the polymer modified porous substrate; applying positive pressureto the larger opening of the pipette to insert the polymer modifiedporous substrate into the pipette tip; and compacting the polymermodified porous substrate. In additional methods for preparing solidphase extraction pipettes, hydrophobic monomers can be polymerized onthe porous substrate after its insertion into the pipette. In certainpreferred embodiments, the polymer modified porous substrate can bepolar functionalized prior to or after insertion into the pipette.

In an additional embodiment, the polymer modified porous substrate canbe used in the form of a disk and incorporated into a solid phaseextraction plate utilizing one or a plurality of solid phase extractiondisks press fitted between the sidewalls of the chambers, as describedin U.S. Pat. No. 5,906,796 to Blevins.

In an additional embodiment, the polymer modified porous substrate isused in a solid phase extraction cartridge or multi-welled plate.Examples of such cartridges (e.g., syringe barrel cartridges) andmulti-well plates are described in, for example, U.S. Pat. No.6,200,533.

VIII. Methods of Preparing Polymer Modified Porous Substrates

The polymer modified porous substrate is formed on the porous substrateutilizing the free radical reaction of hydrophobic monomers comprisingpolymerizable olefin moieties, such as vinyl, allyl, propenyl, butenyl,and the like. The method for preparing a polymer modified poroussubstrate generally comprises (a) contacting a porous substrate with asolution comprising one or more hydrophobic monomers, a porogenicsolvent and a polymerization initiator; and (b) heating the poroussubstrate and retained solution in the absence of oxygen to polymerizethe one or more monomers onto the porous substrate. The method canfurther comprise (c) removing excess solution from the porous substrateprior to heating. In a preferred embodiment, the porous substrate is aglass fiber monolith, and the polymeric monolith formed thereon isformed from hydrophobic monomers that comprise a mixture ofcrosslinkable and uncrosslinkable monomers in a molar ratio of 1:1000 to1000:1, or more preferably 1:100 to 100:1, respectively. However, thereis no particular limit to the amount of crosslinkable monomer oruncrosslinkable monomer that can be used, and in principle 100% ofeither can be utilized with good results.

The hydrophobic monomers preferably comprise aryl moieties includingC₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl groups, optionallysubstituted with nonpolar substituents including C₁₋₁₂ branched orunbranched hydrocarbyl, halo, for example. Preferably, the hydrophobicmonomer has the formulaCH₂═CR-L-A-L_(r)[—CR═CH₂]_(r),where L is a bond or a C₁₋₁₂ branched, unbranched, or cyclichydrocarbyl; A is a C₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl,optionally substituted with C₁₋₁₂ branched or unbranched hydrocarbyl, orhalo; and r is 0 or 1. Typical aryl moieties include benzene, toluene,xylene, and naphthalene, though heteroaryl moieties such as thiophene,thiazole, furan, imidazole, benzimidazole, benzofuran, pyridine,pyrimidine, pyrazole, pyrrole, pyrazine, purine, and the like thatprovide more hydrophilic character can also be utilized. In particularembodiments, the aryl moiety can comprise a bicyclic ring wherein onering is aryl while the other is not aryl, or multiple rings such asfluorene.

Typical monomers are mono- or di-substituted with vinyl, methylvinyl,ethylvinyl, allyl, propenyl, butenyl, etc. Monomers having only onepolymerizable olefin group are uncrosslinkable, and typically includevinyl or allyl substituted C₅₋₁₀ monocyclic or bicyclic aryl orheteroaryl, optionally substituted with C₁₋₁₂ branched or unbranchedhydrocarbyl, or halo, or combinations thereof. Particular examples ofuncrosslinkable monomers include styrene, vinylpyridines (e.g.,2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine), vinylnaphthalene,vinylxylene, allyltoluene, vinylthiophene, vinylthiazole, allylfuran,allylimidazole, vinylbenzimidazole, vinylpyrazole, allylpyrrole,vinylpyrazine, or combinations thereof.

Monomers having at least two polymerizable olefin groups arecrosslinkable, and typically include divinyl or diallyl substitutedC₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl, optionally substitutedwith C₁₋₁₂ branched or unbranched hydrocarbyl, or halo, or combinationsthereof. Particular crosslinkable monomers include divinylbenzene,divinyltoluene, divinylxylene, diallylnaphthalene, divinylpyridine(e.g., 2,4-divinylpyridine), diallylthiophene, divinylthiazole,divinylfuran, diallylimidazole, divinylbenzimidazole, diallylpyridine,divinylpyrazole, diallylpyrrole, divinylpyrazine, or combinationsthereof.

Once formed on the porous substrate, the polymer can be functionalizedwith a polar functionality in order to vary the adsorptioncharacteristics of the polymer. Accordingly, the aryl or heteroarylmoieties, can be functionalized with a polar functionality after thepolymeric monolith is formed on the porous substrate, by treating thearyl or heteroaryl moieties to introduce a polar substituent-L-Q_(p)-R_(q); wherein Q is —NRC(O)—, —C(O)NR—, —OC(O)NR—, —OC(O)R—,—NRC(O)O—, —NRC(O)NR—, —NCO, —CHOHCHOH—, CH₂OCHCH₂O—, —(CH₂CH₂O)_(s)—,—(CH₂CH₂CH₂O)_(s)—, —C(O)—, —C(O)O—, —CH₂C(O)CH₂—, —S—, —SS—, —CHOH—,—O—, —SO—, —SO₂—, —SO₃—, —OSO₃, —SO₂NMe—, —NR_(q)—, and —NR_(q) ⁺—, —CN(nitrile), —NC, —CHOCH—, —NHC(NH)NH—, —NO₂, —NO, —OPO₃—, —OH, where s isgreater than 1, or more preferably 1-12; and R is hydrogen, C₅₋₁₀monocyclic or bicyclic aryl or heteroaryl, C₁₋₁₂ branched, unbranched,or cyclic hydrocarbyl; and wherein q is 0-3; p is 0-5, or morepreferably p is 0-2; and L is a bond or a C₁₋₁₂ branched, unbranched, orcyclic hydrocarbyl. In preferred embodiments, the polar functionalitythat is introduced is amide, nitro, carbamide, urethane, carbamyl,carbamate, carboxyl or hydroxyl, and can also include ion exchangefunctionalities such as amine and sulfonate, or mixtures of any of thepolar functionalities.

An exemplary method for aminating the polymer modified porous substrateis described in Example 2. Briefly, the method involves nitrating thepolymer modified porous substrate to form the nitrated polymer modifiedporous substrate; reducing the nitrated polymer modified poroussubstrate to form the aminated polymer modified porous substrate; andacylating the aminated polymer modified porous substrate with an acid,an acid chloride or an acid anhydride to form the amidated polymermodified porous substrate. Generally, the nitrating comprises: (a)contacting the polymer modified porous substrate with a first solutioncomprising nitric acid; and (b) contacting the polymer modified poroussubstrate with a second solution comprising a reagent adapted togenerate a nitronium ion to the first solution. In a preferred method,the procedure involves treating the polymer modified porous substratewith concentrated nitric acid (about 30 molar equivalents) withstirring. While cooling the mixture in cold water, concentrated sulfuricacid (18 molar equivalents) is added dropwise over a period of 1 to 1.5hours, continuing the stirring. The nitrated polymer modified poroussubstrate can then be removed and washed with water, 2.0M NaOH, waterand acetone, and dried.

The nitrated polymer modified porous substrate is then subjected to areduction step, wherein the reducing comprises: contacting the nitratedpolymer modified porous substrate with a first solution comprising afirst acid; and contacting the nitrated polymer modified poroussubstrate with a second solution comprising a metal catalyst and asecond acid. Preferably, the first acid is an organic acid, and thesecond acid is selected from hydrochloric acid, an organic acid orcombinations thereof. Preferably, the metal catalyst is selected fromstannous chloride, zinc metal, an organo-metallic hydride, or hydrogenin the presence of a metal. In a preferred method, the procedureinvolves: suspending and stirring the nitrated polymer modified poroussubstrate in glacial acetic acid at room temperature, while treatingwith a solution of stannous chloride in 1:1 hydrochloric acid. Theaminated polymer modified porous substrate is removed from the reactionand washed with water, and 1.0 M sodium hydroxide until all traces oftin are washed out. Then the aminated polymer modified porous substrateis washed with water until the wash water pH is neutral, and then iswashed with acetone, and dried.

The aminated polymer modified porous substrate can then be furthermodified with an additional moiety if desired, such as hydrocarbyl, arylor heteroaryl, for example, using alkylating agents such as iodomethane,iodoethane, iodotoluene, or iodofuran to prepare secondary, tertiary orquaternary amines. Alternatively, the amine functionalized polymermodified porous substrate can be further treated to result in an amidepolar functionality by acylating the aminated polymer with a suitablereactant, such as an acid, acid chloride or anhydride. Typically, theacylating comprises: suspending the reduced polymer modified poroussubstrate in a first solution comprising a base to form a basic reactionsolution; and adding an acid, an acid chloride or an anhydride to thebasic reaction solution. Preferably, the base is selected fromtriethylamine, pyridine, alkyl pyridines, quinoline, alkylquinolines,trialkylamines, imidazole or triazole. Preferably, the acid chloride isselected from acetyl chloride, alkanoyl chlorides, aryl chlorides andheteroaryl chlorides; the anhydride is selected from acetic anhydride,anhydrides of aliphatic acids (C₂₋₁₂), anhydrides of aromatic acids,anhydrides of heterocyclic acids; and the acid is selected fromaliphatic acids, aromatic acids, or heterocyclic carboxylic acids.Mixtures of any of these acylating agents can also be utilized.

An exemplary method for introducing an amide moiety is described inExample 2. The aminated polymer modified porous substrate is suspendedin excess base (e.g., triethylamine or pyridine), and with stirring, istreated dropwise with the acid, acid chloride or anhydride (1.5 moleequivalent to mole of nitrogen of the aminated polymer), at roomtemperature. The amide functionalized polymer modified porous substrateis removed from the reaction mixture and washed several times with 0.1Mhydrochloric acid and then with deionized water, methanol and acetone.Finally, the amide functionalized polymer modified porous substrate isdried. Preferably, the amide functionalized polymer modified poroussubstrate comprises between about 0.1% and about 10.0% nitrogen by masspercent, or more typically, between about 0.4% and about 5.0% nitrogen.The polar functionalized polymer modified porous substrate remainssolvated after contact with water and an organic solvent for longer thanabout one hour.

The polymer modified porous substrates can also be functionalized withion exchange functionalities to provide ion exchange capabilities. Apreferred cation exchange functionality is sulfonate, which can beintroduced onto the polymer modified porous substrate by sulfonation. Anexemplary method for sulfonating the polymer is described in U.S. Pat.No. 6,322,695 to Lee. Briefly, a sulfonated polymer modified poroussubstrate (e.g., poly(styrenedivinylbenzene) orpoly(divinylbenzene-co-N-vinylpyrrolidone) copolymer modified glassfiber) can be prepared by derivatization with sulfuric acid as follows:Sulfuric acid and the polymer modified porous substrate is introducedinto a flask fitted with a thermometer, agitator, condenser and reactortemperature control system to a weight ratio of about 5 to 100H₂SO₄(95-98%) to polymer, and reacted at a chosen temperature and agitationadjusted to ensure adequate mixing. The reaction mixture is stirred fora certain period of time at constant temperature, and then the polymermodified porous substrate is washed with water, and then dried. Thelevel of sulfonation can be determined by sulfur elemental analysis(e.g., Atlantic Microlab Inc., Norcross, Ga.). The reaction temperatureand reaction time can be chosen to prepare polymers with desired ionexchange capacities.

A preferred anion exchange functionality is quaternary amino, which canbe introduced onto the polymer modified porous substrate by aminationand alkylation, as mentioned above. Briefly, the procedure described byCorradini, C. et al. (1994) J. Chromatography A Vol. 685, pp. 213-220can be utilized as follows: the polymeric monolith formed on the poroussubstrate can be nitrated by adding the polymer modified poroussubstrate to a cold solution of 75% (v/v) fuming nitric acid andsulfuric acid and stirred for 3 hours, and then heated to 60° C. for 6hours. The nitrated polymer modified porous substrate can be washed withwater, sodium hydroxide, water and solvent and dried. The nitratedpolymer modified porous substrate is then reduced in dimethylformamideusing granulated tin metal and hydrochloric acid at 5° C. followed byheating to 25° C. for 2 hours, followed by heating to 90° C. andmaintaining this temperature overnight. The aminated polymer modifiedporous substrate can then be washed with water, sodium hydroxide, waterand solvent and dried. Finally, the aminated polymer modified poroussubstrate can be quaternized by methylation using iodomethane (excess)and heating to 60° C. for 10 hours. After reaction, the polarfunctionalized polymer modified porous substrate can be washed withwater, solvents, triethylamine, solvents and dried.

One skilled in the art will readily envision additional methods forintroducing polar functionalities that can be provided on the polymermodified porous substrate. For example, nitrile functionalized polymermodified porous substrates can be provided by reaction ofbenezenesulfonic acid moieties (produced as described above) with KCN,or by dehydration of amide functionalized polymer modified poroussubstrates. Hydroxyl functionalized polymer modified porous substratescan be provided by reaction of benzenesulfonic acid moieties (producedas described above) with caustic alkali (NaOH, KOH). Carboxyfunctionalized polymer modified porous substrates can be provided byreaction of a polymer modified porous substrate comprising alkylsubstituted aryl or heteroaryl (e.g., toluene) with KMnO₄. Carbamidefunctionalized polymer modified porous substrates can be provided byreaction of aminated polymer with ClC(O)NR. Carbamyl functionalizedpolymer modified porous substrates can be provided by reaction ofcarboxy functionalized polymer with NR₃, where R is hydrogen,hydrocarbyl, aryl or heteroaryl. Alkyloxy functionalized polymermodified porous substrates can be provided by reaction of hydroxylfunctionalized polymer with an acid, acid chloride or anhydride (e.g.,acetoxy can be prepared from ClC(O)CH₃). Carbamate functionalizedpolymer modified porous substrates can be provided by reaction ofaminated polymer with chloroformates. Urethane functionalized polymermodified porous substrates can be provided by reaction of hydroxylfunctionalized polymer with an isocyanate. Isocyanate functionalizedpolymer modified porous substrates can be provided by reaction ofaminated polymer with phosgene, for example. Isonitrile functionalizedpolymer modified porous substrates can be provided by reaction ofaminated polymer with chloroform.

One skilled in the art will recognize that the amount of polarfunctionality can be controlled and chosen to provide the desiredadsorption of analytes. For example, as discussed in Example 3, thePSDVB—NHCOCH₃ modified porous substrate contains 2.55% nitrogen, butlesser or greater amounts of polar functionality could be utilized toprovide a PSDVB—NHCOCH₃ having lesser or greater amounts of nitrogen toprovide desired adsorption characteristics. Accordingly, the presentmethods for preparing polar functionalized polymer modified poroussubstrates also provide methods for controlling the adsorption ofanalytes having a particular polarity, and for tailoring the polymermodified porous substrate to achieve that goal.

In an additional embodiment, polar functionalized polymer modifiedporous substrates can be prepared comprising mixtures of polarfunctional groups. In this embodiment, the polymer modified poroussubstrates can provide both superior retention of polar analytes,superior wettability, and specific adsorption capabilities, such as ionexchange functionality. In preferred embodiments, the mixtures of polarfunctional groups include amide+amine, amide+sulfonic acid,nitrile+sulfonic acid, and amide+quaternary amine. However, any of thepolar groups can be used as mixtures.

IX. Methods for Separating Analytes

The polymer modified porous substrates can be utilized in methods ofseparating compounds for analysis, such as chromatography. Suitablechromatographic applications or separation methods include, for example,thin layer chromatography, high performance liquid chromatography,reversed phase chromatography, normal phase chromatography, ionchromatography, ion pair chromatography, reverse phase ion pairchromatography, ion exchange chromatography, affinity chromatography,hydrophobic interaction chromatography, size exclusion chromatography,chiral recognition chromatography, perfusion chromatography,electrochromatography, partition chromatography, microcolumn liquidchromatography, capillary chromatography, liquid-solid chromatography,preparative chromatography, hydrophilic interaction chromatography,supercritical fluid chromatography, precipitation liquid chromatography,bonded phase chromatography, fast liquid chromatography, flashchromatography, liquid chromatography-mass spectrometry, gaschromatography, microfluidics based separations, solid phase extractionseparations, or monolith based separations, without limitation. Apreferred chromatographic application for the polymer modified poroussubstrates is solid phase extraction, for example, performed using a SPEcartridge, or ion exchange chromatography.

The miniaturization of liquid separation techniques to the nano-scaleinvolves small column internal diameters (<100 micron i.d.) and lowmobile phase flow rates (<300 mL/min). Techniques such as capillarychromatography, capillary zone electrophoresis (CZE), nano-LC, opentubular liquid chromatography (OTLC), and capillaryelectrochromatography (CEC) offer numerous advantages over conventionalscale high performance liquid chromatography (HPLC). These advantagesinclude higher separation efficiencies, high-speed separations, analysisof low volume samples, and the coupling of 2-dimensional techniques.

A method for performing a chromatographic separation of analytes cangenerally comprise a) providing a polymer modified porous substratedisposed in a chromatography apparatus (e.g., column, cartridge,channels on a microchip, etc.); b) conditioning said polymer modifiedporous substrate with one or more mobile phases (e.g., an organicsolvent, aqueous solution of optional buffers or salts, or mixturesthereof, such as acetonitrile/formic acid/water, or gradients of solventmixtures); c) contacting said polymer modified porous substrate with asolution comprising one or more analytes (e.g., plasma, environmentalwater sample, mixtures of synthetic reactants, products andintermediates, etc.); d) passing a mobile phase comprising an organicsolvent, an aqueous solution, or mixtures thereof, through said polymermodified porous substrate; and e) eluting one or more analytes from thepolymer modified porous substrate (e.g., using an appropriate mobilephase comprising counterions, organic solvents, etc.). As the polymer isformed directly on the porous substrate, channeling and shrinkage of thesorbent (the polymer modified porous substrate) within thechromatography apparatus can be minimized when using a nonswellableporous substrate such as a glass fiber monolith.

X. Methods for Performing Solid Phase Extraction

In a particular aspect, a method for isolating an analyte from a sampleusing solid phase extraction is disclosed. Representative procedures forperforming solid phase extraction are described in Examples 4-6. Thesample can be derived from any source, although the polymer modifiedporous substrate and methods described herein are particularly suitedfor isolating an analyte from biological, environmental, synthetic andpharmaceutical samples, and the like. For example, a sample can comprisea biological matrix (e.g., whole blood or plasma or saliva or urine,etc.) comprising an analyte of interest (e.g., a drug, peptide orhormone, etc.). Alternatively, a sample can comprise an environmentalsample, such as an extract of soil or, or a drinking water sample orwater known or suspected of being polluted (e.g., effluent water). Inanother example, the sample can comprise an analyte that is atherapeutically-active agent or precursor or metabolite thereof.

The method for isolating an analyte generally comprises four steps: (a)conditioning a polymer modified porous substrate with an organic solventand optionally an aqueous solvent, or mixtures thereof; (b) adsorbinganalytes present in a sample to be analyzed to the conditioned polymermodified porous substrate; and (c) eluting the adsorbed analytes fromthe polymer modified porous substrate with an organic solvent, anaqueous solvent, or mixtures thereof. The method can further comprisewashing the polymer modified porous substrate and adsorbed analytesprior to performing step (c), and can further comprise analyzing theanalyte using any suitable analytical methodology or instrumentation.

The conditioning step generally comprises treating the polymer modifiedporous substrate with solvents that enhance surface characteristics. Ina preferred embodiment, the method comprises conditioning the polymermodified porous substrate by washing the polymer modified poroussubstrate with an organic conditioning solvent (e.g., methanol) followedby water, for example about 1 mL each. The polymer modified poroussubstrate can be associated with a support, such as a cartridge orcolumn, in which case, the step of conditioning can comprise passing anorganic solvent over the cartridge or column, followed by passing anaqueous solvent over the cartridge or column. The methanol swells thepolymer modified porous substrate and enhances the effective surfacearea. The water treatment removes excess methanol and hydrates thepolymer surface. The conditioned surface can then be subjected to vacuumto remove excess solvents; the polymer modified porous substrate remainscompletely hydrated after this treatment. A sample comprising an analytecan then be contacted with the polymer modified porous substrate to forma polymer modified porous substrate-analyte complex. This step,sometimes referred to as sample loading, allows adsorption of one ormore analytes, if present, on the polymer modified porous substrate. Theadsorption of analytes, and hence their retention, is a function of thehydrophobic as well as hydrophilic (e.g., dipolar, proton accepting,proton donating, electrostatic attraction, etc.) interactions ofanalytes with the polymeric sorbent formed on the porous substrate.Thus, a polymer modified porous substrate that facilitates hydrophobicas well as hydrophilic interactions with analytes will retain analytesof varying polarities and chemical characteristics. Desorption andrecovery of the analytes can then be performed using a solvent that iscapable of disrupting the hydrophobic and hydrophilic interactions ofthe polymeric sorbent with the analytes.

Accordingly, in a preferred embodiment, the polymer modified poroussubstrate is used in a solid phase extraction format and comprises apolymer modified porous substrate that has been polar functionalized.Exemplary polymeric monoliths formed on the porous substrate includepoly(styrene-divinylbenzene), copolymers formed from styrene, allylbenzene, divinyl or diallylbenzene, hydrocarbyl or halo substitutedstyrenes, vinylfurans, vinylpyridines, etc. and combinations thereof,and is polar functionalized with amide, which provides a very favorableretention of polar analytes and a linear correlation of retention withanalyte polarity. In additional preferred embodiments, the polymermodified porous substrate is functionalized with other polar groups suchas amino, quaternary amino, nitro, sulfonic acid, and the like.

When a sample comprises blood plasma, the sample can be introduced asdiluted aqueous solutions (at least 1:1 dilution). This practice can bedesirable because of the high viscosity of plasma samples from animalsor humans, which prevents free flow unless diluted to reduce viscosity.However, due to the excellent flow properties of the polymer modifiedporous substrates, sample viscosity is less problematic than withconventional sorbents used for solid phase extraction. It is usuallypreferable to avoid the use of organic solvents in this step, sincethese solvents can precipitate proteins from the plasma solution and theprecipitated proteins can foul the sorbent surface. Again, the excellentflow properties of the polymer modified porous substrates obviate someof this difficulty. It can also be desirable that a sample is contactedwith the polymer modified porous substrate under conditions conducive toadsorption of the analyte and the polymer. At the same time, theseconditions are preferably unfavorable for retaining unwanted proteinsand other impurities on the sorbent surface. Such conditions can includeconducting the contacting at about room temperature and neutral pH.

In a one embodiment, a sample is loaded in a 1:1 aqueous solution and ananalyte (e.g. a drug) can be present in one nanogram to 10 microgram permilliliter levels. A sample volume of about 100 to about 1000microliters can be loaded, although volumes of about 400 to about 500microliters are preferred. The polymer modified porous substrate-samplecomplex can then be washed with water, followed by an organic washsolvent. In one embodiment, the sample loaded sorbent is washed withwater and then with about 10 to about 30% acetonitrile in water (anyvolume can be employed, although volumes from about 200 to about 1000microliters are preferred). The water wash removes salts and otherwater-soluble matrix constituents that might be present in a sample, inaddition to proteinaceous matter. The binary aqueous-organic wash canalso remove organic impurities including water-insoluble matrixcomponents that can adhere to the sorbent surface. It can be desirableto configure this wash so as not to disrupt the binding of an analyte tothe polymeric sorbent surface. When many known silica based andpolymeric sorbents are employed in a separation, such a binary wash canremove many polar analytes from the sorbent.

Next, an analyte can be eluted from the polymer modified poroussubstrate-analyte complex with an eluting solvent. The elution can beperformed by passing a volume of an eluting solvent over the polymermodified porous substrate having adsorbed analytes. Representativeeluting solvents include binary solvents comprising an aqueous componentand an organic component. Preferably, the organic component comprises atleast about 80-90% of the solvent. Representative organic componentsinclude, but are not limited to, acetonitrile and methanol. A trailingion, such as trifluoroacetic acid, can also be employed as a componentof an elution solvent and serves to disrupt the polar interactions ofpolar drugs with the sorbent effectively. In one embodiment, a 60:30:10methanol/acetonitrile/0.1% trifluoroacetic acid is found to afford 90%to almost quantitative recoveries of drugs of a wide range of polarities(see FIG. 6). Eluting solvent volumes of about 400 microliters to about1000 microliters can be employed, and volumes about 400 to about 500microliters are preferred in some situations.

The eluent can be collected and the identity the recovered analytesascertained, for example by mass spectrometry, liquid chromatography,gas chromatography or a combination of these and other techniques knownto those of ordinary skill in the art. When an analyte of interest (e.g.a drug) is present in picogram levels in plasma, the eluting solvent canbe evaporated and the residual analyte redissolved (i.e. reconstituted)in about 40 to about 100 microliters of the mobile phase used for LC orLC/MS.

An advantage of the polymeric sorbents and associated methods of thepresent invention is the ability to pass eluent directly to aninstrument(s) for analyte identification. The direct passage toanalytical instrumentation is not possible with many prior art sorbents,due, in part, to ion suppression effects of prior art sorbents and theinability of these sorbents to retain moderately polar to highly polaranalytes. These deficiencies can lead to unwanted components in aneluent, which can significantly complicate analyte identificationoperations, and poor MS spectra. For example, a sorbent of the presentinvention can form a component of a system comprising the sorbent and aLC/MS/MS system. Samples can be loaded onto the sorbent, analytes elutedand the eluent stream fed directly into an LC/MS/MS system, HPLC systemor any of a range of analytical instruments.

XI. Advantages of the Polymer Modified Porous Substrates

The polymer modified porous substrates described herein demonstratesuperior flow rates, as demonstrated in Example 7, providing for fastersample preparation times and increased efficiencies in samplepreparation. The polymer modified porous substrates exhibit flow ratesof at least 40 mL/min, between about 40 mL/min to about 1 L/min, andmore typically at least 100 mL/min, when tested using a solventcomprising 5% methanol in water through a disk having a diameter ofabout 47 mm. The superior flow characteristics result in reduced oreliminated channeling effects, as well as decreased clogging of theporous substrates during extraction of analytes, providing for greaterconsistency in analyte recovery and additional savings in time andexpenses.

The polymer modified porous substrates described herein provide improvedanalyte recoveries, and recovery efficiencies. The substrates describedherein are especially advantageous when polar functionalized and usedwith samples containing polar analytes, which can be challenging torecover for analysis. In addition, the substrates described herein canbe used in high throughput applications, providing for highproductivities.

Further, analytes can be eluted from the sorbents using small elutionvolumes, providing for more highly concentrated samples for analysis,decreased solvent volumes, elimination of a solvent evaporation step,and subsequent time savings. These features provide for savings in timeand labor as well as materials.

The polymer modified porous substrates described herein provide varyingretention of analytes having varying polarities, which providesadvantages in isolating analytes of desired polarities. The polarfunctionalized polymer modified porous substrates provided hereinexhibit improved retention of polar analytes, and superior retention ofpolar analytes relative to a polymeric monolith that is not polarfunctionalized. The polar functionalized polymer modified poroussubstrates also provide superior adsorption capacity for polar analytes.The polymer modified porous substrates provide a linear correlationbetween analyte retention and log P across a range of log P values, andthe polar functionalized polymer modified porous substrates provide alinear correlation for across a broader range of log P values. Incertain embodiments, the improved retention of polar analytes can beprovided without the use of an ion exchange functionality.

As shown in Example 5, retention of each analyte by the polymer modifiedporous substrate, PSDVB modified glass fiber, was linear across a rangeof log P values from about 2.5 to 5. However, below log P values of 2.5,the retention was reduced, with relative retention of less than 80% foranalytes having log P values of about 1.75, and less than 60% foranalytes having log P values of about 1.6. Thus retention of relativelynonpolar analytes (e.g., analytes having log P values of greater than2.5) by PSDVB modified porous substrates is preferred

The relative retention of analytes by the polar functionalized polymermodified porous substrate, PSDVB—NHCOCH₃ modified glass fiber, waslinear across the range of log P values tested, from about 1.6 to 5,with relative retention of most analytes being within 90% of theretention of nortriptyline. Thus retention of analytes is less dependenton analyte polarity with the polar functionalized polymer modifiedporous substrate.

Use of the polymer modified porous substrates allows recovery ofadsorbed analytes across a wide range of log P values in a small volumeof elution solvent, minimizing the need to concentrate samples forfurther analysis. The elution of analytes from the PSDVB modified glassfiber indicates that the polymer modified porous substrate adsorbsanalytes across a wide range of polarities, with similar recoveriesobserved for polar compounds (ephedrine) and nonpolar compounds(quinidine) (FIG. 4). Other analytes are retained longer, but could beeluted with similar solvent volumes. Thus, for certain analytes,recovery varies with solvent volume, showing some separation effect withrecoveries of 75% to 95% of adsorbed analytes recovered after elutingwith 400 μL or more of eluting solvent.

As shown in FIG. 5, the elution of analytes from PSDVB—NHCOCH₃ modifiedglass fiber demonstrated very good recoveries of analytes having a widevariety of polarities using low volumes of eluting solvent. Greater than95% recoveries were observed for all analytes with 400 μL of elutingsolvent.

The capacity of the polymer modified porous substrates for analytes ishigh, and can be selected to retain analytes of a desired polarity. Thenonpolar polymer surfaces retain nonpolar analytes preferentially, butwhen polar functionalized, also retain polar analytes as well. As shownin Example 6, the capacity of PSDVB—NHCOCH₃ modified porous substratesfor polar analytes was generally about 1-2 μg retained. Where greatercapacity is desired, additional polymer modified porous substrate can beutilized, for example, by employing a greater volume of polymer modifiedporous substrate, or adding additional devices. In a preferredembodiment, the polymer modified porous substrate is in the form of aglass fiber disk, and additional disks can be stacked to provide agreater analyte adsorption capacity.

In addition, the methods of preparing the polar functionalized polymermodified porous substrates described herein are efficient and simple,providing savings in cost and labor in the manufacturing process. Thepolar functionalized polymer modified porous substrates described hereinare also efficient and simple to use, due to the high retention ofanalytes of varying polarities as well as the fast solvent flow rates,providing savings in time and solvent requirements for analysis ofanalytes.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that thedescription above as well as the examples that follow are intended toillustrate and not limit the scope of the invention. The practice of thepresent invention will employ, unless otherwise indicated, conventionaltechniques of organic chemistry, polymer chemistry, biochemistry and thelike, which are within the skill of the art. Other aspects, advantagesand modifications within the scope of the invention will be apparent tothose skilled in the art to which the invention pertains. Suchtechniques are explained fully in the literature.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in ° C. and pressure is at or nearatmospheric. All solvents were purchased as HPLC grade, and allreactions were routinely conducted under an inert atmosphere of argonunless otherwise indicated. Unless otherwise indicated, the reagentsused were obtained from the following sources: dodecanol, THF, styrene,DVB, AIBN, and decanol were obtained from Sigma Aldrich.

Abbreviations: DVB divinylbenzene AIBN azobisisobutyronitrile THFtetrahydrofuran ST styrene

Example 1 Preparation of a Polymer Modified Porous Substrate

A PSDVB modified glass fiber monolith was prepared by the followingprocedures. The reaction is depicted in schematic fashion in FIG. 1.

A polymer forming solution was prepared containing styrene (10 mL),divinylbenzene (10 mL), 1-decanol (26 mL), tetrahydrofuran (4 mL) andazobisisobutyronitrile (200 mg) (AIBN) and stirred in a beaker untildissolved, then the mixture was poured into a wide container (a plastictray). The mass of an approximately 4.5″×3.0″ piece of glass fiber mediawas determined and recorded, and then immersed into the above preparedmonomer mixture for about 7 minutes. The saturated glass fiber wasremoved from the tray and allowed to drip for about 1.5 minutes toremove any excess reaction mixture. The solution saturated glass fiberwas placed into an appropriate container (a vented desiccator withoutdesiccant) having a means to support the glass fiber. The container waspurged with dry nitrogen for about 30 minutes to remove air from thedesiccator. The purge line was removed and the desiccator vent wasclosed. The desiccator was then placed in an oven at 70° C. for 24 hoursfor the polymerization reaction. After the reaction period, the polymermodified glass fiber monolith (“PSDVB”) was removed and extensivelywashed with methanol, water and acetone and then dried in an oven at 70°C. for 24 hours.

Example 2 Preparation of an Amide Functionalized Polymer Modified PorousSubstrate

A PSDVB modified glass fiber monolith was amidated by the followingprocedures. The reaction is depicted in schematic fashion in FIG. 2.

A. A nitration step was performed as follows: the PSDVB modified glassfiber monolith prepared in Example 1 was suspended in concentratednitric acid (30 molar equivalents) and the mixture was mechanicallystirred. While cooling the mixture in cold water, concentrated sulfuricacid (18 molar equivalents) was added dropwise over a period of 1 to 1.5hours, continuing the stirring at the same time. The mixture was furtherstirred at room temperature for three more hours. The modified glassfiber monolith was removed and immersed in water, and allowed to standfor a couple of hours, and was then washed with 2.0M NaOH, deionizedwater and acetone, and dried in an oven at 70° C. for 24 hours.

B. A reduction step was performed as follows: the nitrated PSDVB glassfiber monolith was suspended in glacial acetic acid, and while beingmechanically stirred, was treated with a solution of stannous chloride(375 g) in 1:1 hydrochloric acid (951 mL). The mixture was stirred atroom temperature for 60 hours. The polymer modified glass fiber monolithwas removed from the reaction and immersed in deionized water, andwashed with 1.0 M sodium hydroxide several times until all traces of tinwere washed out. Then the monolith was washed with water until the washwater pH was neutral, and then was washed with acetone. The monolith wasthen dried in an oven at 70° C. for 24 hours.

C. An acylation step was performed at follows: The aminated PSDVB glassfiber monolith was suspended in a base (triethylamine or pyridine,excess) and with slow mechanical stirring, was treated dropwise withacetic anhydride (1.5 mole equivalent to mole of nitrogen of theaminated polymer). The stirring was continued for 3.5 hours at roomtemperature. The amide functionalized polymer modified glass fibermonolith (“PSDVB—NHCOCH₃”) was removed from the reaction mixture andwashed several times with 0.1M hydrochloric acid and then with deionizedwater, methanol and acetone. Finally, the monolith was dried at 70° C.

Example 3 Elemental Analysis of Polymer Modified Monoliths

The amide functionalized polymer modified glass fiber monolith describedin Example 2 PSDVB—NHCOCH₃, and the PSDVB modified glass fiber monolithprepared according to Example 1 (i.e., prior to the nitration, reductionand acetylation steps), were subjected to elemental analysis. Elementalanalysis for the amide functionalized polymer modified glass fibermonolith, PSDVB—NHCOCH₃, showed 48.37% carbon, 4.02% hydrogen and 2.55%nitrogen, with the remaining composition being silicates. The glassfiber monolith modified with PSDVB alone had negligible nitrogen content(N<0.02%).

Example 4 Relative Retention of Analytes by Polymer Modified Glass FiberMonolith

A solution was prepared in 2% methanol containing the following mixtureof analytes. The final concentration of each analyte was amphetamine(2500 ng/mL), ephedrine (5900 ng/mL), nortriptyline (2280 ng/mL),mianserin (2170 ng/mL), chlorpheniramine maleate (4500 ng/mL),norfluoxetine (4000 ng/mL), brompheniramine (3000 ng/mL), and quinidine(4400 ng/mL). The log P values of each analyte are as shown in Table 1below.

TABLE 1 Log P values for various analytes Analyte Log P Amphetamine 1.76Ephedrine 1.61 Nortriptyline 4.74 Mianserin 3.85 Chlorpheniraminemaleate 3.38 Norfluoxetine 3.75 Brompheniramine 3.89 Quinidine 2.62

Ninety-six well solid phase extraction plates were prepared with eitherPSDVB—NHCOCH₃ or PSDVB. Each sorbent was conditioned with 400 μLmethanol and 400 μL deionized water. Then 200 μL of analyte mixture wasapplied to the sorbent in each well. Analytes were eluted from eachsorbent using 2×100 μL aliquots of 6:3:1 methanol/acetonitrile/1% formicacid (200 μL total). A 50 μL aliquot of the pooled eluant was dilutedwith an additional 50 μL 1% formic acid (total volume 100 μL), and 10 μLof the diluted eluant was analyzed and quantitated by LC-MS (Varian1200L mass spectrometer) using a PURSUIT® C18 column (50×2.0 mm, 3 μmparticle size) with the mobile phase gradient described in the tablebelow. N=5 for each sorbent.

TABLE 2 Mobile phase gradient % Formic acid Flow Rate Time % Methanol(0.1%) (μL/min) 0:00 15 85 300 0:30 15 85 300 2:00 85 15 300 2:30 85 15300 2:31 15 85 300 3:30 15 85 300

The retention of each analyte was plotted as a function of log P for theanalyte versus % recovery from the sorbent. The retention of analytes bythe PSDVB modified glass fiber monolith alone is linear across a rangeof log P values from about 2.5 to 5, and about 67% of each analytehaving log P values in this range was recovered in the elutionfractions. However, below log P values of 2.5, the recovery was reduced,with recoveries of 51% for the analyte having a log P value of 1.76, and40% for the analyte having a log P value of 1.6, indicating thatanalytes having low log P values are retained as well as analytes havinghigher log P values by the hydrophobic PSDVB modified glass fibermonolith. In contrast, the retention of analytes by the PSDVB—NHCOCH₃modified glass fiber monolith of Example 2 was linear across a range oflog P values from about 1.6 to 5, and 63% to 73% of each analyte havinglog P values in this range was recovered.

As shown in FIG. 3, when normalized to nortriptyline and expressed asrelative retention, the retention of each analyte by PSDVB modifiedglass fiber monolith was linear across a range of log P values fromabout 2.5 to 5. Again, below log P values of 2.5, the retention wasreduced, with relative retention of less than 80% for analytes havinglog P values of about 1.75, and less than 60% for analytes having log Pvalues of about 1.6. In contrast, the relative retention of analytes bythe PSDVB—NHCOCH₃ modified glass fiber monolith was linear across arange of log P values from about 1.6 to 5, with relative retention ofmost analytes being within 90% of the retention of nortriptyline.

Example 5 Capacity of Polymer Modified Glass Fiber Monoliths forAnalytes

Stock solutions were prepared containing the following mixture of polaranalytes at three different concentrations: high, intermediate and low.The concentrations of analyte in each stock solution was as follows:atenolol (840, 42, 2 μg/mL), ranitidine HCl (806, 40, 2 μg/mL),salbutamol (810, 40, 2 μg/mL), bamathan hemisulfate (806, 40, 2 μg/mL),timolol maleate (823, 41, 2 μg/mL), and carisoprodol (840, 42, 2 μg/mL)at the three different concentrations, respectively. The log P values ofeach analyte are as shown in Table 3 below.

TABLE 3 Log P values for various analytes Analyte Log P Atenolol 0.16Ranitidine HCl 0.27 Salbutamol 0.97 Bamathan hemisulfate 1.29 Timololmaleate 1.83 Carisoprodol 1.96

Ninety-six well solid phase extraction plates were prepared with one ofthe two sorbents, PSDVB—NHCOCH₃ and PSDVB. Each sorbent was conditionedwith 1 mL methanol and 1 mL deionized water. Then 500 μL of analytesolution was applied to the sorbent in each well. The eluant wascollected. Another 500 μL was applied, followed by collection of theeluant. A 1500 μL aliquot was then applied to the sorbent, followed bycollection of the eluant. A final 1500 μL aliquot was applied to thesorbents, and eluant was collected.

The analytes recovered in the eluates from each sorbent at eachconcentration applied (the analytes not adsorbed) were quantitated byLC-MS (Varian 1200 L) using a PURSUIT® C18 column (50×2.0 mm, 3 μmparticle size) using a mobile phase isocratic method with 40%methanol/60% 0.1% formic acid. N=3 for each sorbent and concentration.

The capacity of each sorbent for analytes of varying polarity ispresented in Table 4 below. The sorbent capacity is expressed for eachsorbent as the weight of each analyte adsorbed at 30% breakthrough(i.e., when 30% of the applied analyte elutes through the sorbentwithout being adsorbed).

TABLE 4 Amount of analytes adsorbed by various polymer modified poroussubstrates PSDVB- Analyte Log P PSDVB NHCOCH₃ Atenolol 0.16 0 0Ranitidine 0.27 0 1.1 Salbutamol 0.97 0 0 Bamathan 1.29 0 2.0 Timolol1.83 0 1.2 Carisoprodol 1.96 0 1.2

As presented in Table 4, the capacity of the PSDVB modified glass fibermonolith alone for polar analytes such as timolol and atenolol was <1μg, and no adsorbed analytes were detected, indicating that polaranalytes are not well retained by this hydrophobic sorbent.

The capacity of the PSDVB—NHCOCH₃ modified glass fiber monolith forpolar analytes was determined and small amounts of ranitidine, bamathan,timolol and carisprodol retained (1-2 μg).

Example 6 Comparative Analysis of Elution Characteristics from PSDVBModified Porous Substrates

The recovery of various analytes from two different polymer modifiedglass fiber monoliths was investigated. A mixture of analytes wasprepared containing ephedrine, nortriptyline, mianserin,chlorpheniramine maleate, brompheniramine, and quinidine. Each analytewas at a final concentration of 0.1 μg/mL.

Ninety-six well solid phase extraction plates were prepared withPSDVB—NHCOCH₃ or PSDVB. Each sorbent was conditioned first with 1 mLmethanol, and then with 1 mL deionized water. Then 0.4 mL of the mixtureof analytes was applied to each monolith, and the eluant discarded.Analytes were eluted in fractions from each sorbent at total elutionvolumes of 25 μL, 50 μL, 100 μL, 200 μL, and 1000 μL 6:3:1methanol/acetonitrile/1% formic acid for all sorbents. All fractionswere evaporated and reconstituted in 100 μL of 15% methanol, remainder0.1% formic acid solution.

The quantitation of each analyte eluted was determined by LC-MS (Varian1200 L) using a PURSUIT® C18 column (50×2.0 mm, 3 μm particle size)using the mobile phase gradient described in Table 3 above. N=5 for eachsorbent.

As shown in FIG. 4, the elution of analytes from a PSDVB modified glassfiber monolith indicates that the sorbent adsorbs analytes across a widerange of polarities, with similar recoveries observed for polarcompounds (ephedrine) and nonpolar compounds (quinidine). Other analyteswere retained longer, but eluted with similar solvent volumes. However,the rate of recovery of the analytes showed variability. Larger volumesof eluting solvent were required to elute certain analytes, and recoverywas variable, with 75% to 95% of adsorbed analytes recovered aftereluting with 400 μL of eluting solvent.

As shown in FIG. 5, the elution of analytes from PSDVB—NHCOCH₃ modifiedglass fiber monoliths demonstrated very good recoveries of analyteshaving a wide variety of polarities using low volumes of elutingsolvent. Greater than 95% recoveries were observed for all analytes with400 μL of eluting solvent.

Example 7 Comparison of Flow Characteristics from PSDVB ModifiedMonoliths and a Commercial Solid Phase Extraction Filter Disk

Polymer modified porous substrates were prepared as described in Example1, using varying amounts of divinylbenzene and styrene, as indicated inTable 5 below. A commercial solid phase extraction disk, Empore™ HighPerformance Extraction Disks, SDB-XC, Part #12145010, was purchased andused as a comparator.

All disks were the same size (47 mm in diameter). Flow rates weredetermined by placing the disk into the disk holder screen side down,and the vacuum level was set at 10 inches of mercury. A valve was placedbetween the disk holder and vacuum source. The sample volume was 1 L ofdeionized water containing 5 mL methanol. To perform the test, about 3mL of methanol was added to the disk to condition it prior to sampleaddition. The sample was added to the funnel of the disk holder, vacuumwas applied and a timer started to record the time necessary to draw thesample through the disk. The results of the tests are shown in Table 5below.

TABLE 5 Flow properties of various polymer modified porous substratesDVB ST Decanol Dodecanol THF Flow rate mL mL mL mL mL mL/min PSDVB 15  526 — 4 229 10 10 26 — 4 414  5 15 26 — 4 444 Empore ™ High PerformanceExtraction Disks, SDB-XC — — — — —  28

These results demonstrate the superior flow rates achievable using thepolymer modified porous substrates described herein. The superior flowrates provide for faster sample preparation times and increasedefficiencies.

1. A polymer modified porous substrate for solid phase extraction orchromatography, comprising a porous substrate and a polymeric monolithformed thereon, wherein the polymeric monolith has the formula

wherein A is selected from C₅₋₁₀ monocyclic or bicyclic aryl orheteroaryl, optionally substituted with -L-Q_(p)-R_(q); q is 0-3; p is0-5; Q is —NRC(O)—, —C(O)NR—, —OC(O)NR—, —OC(O)R, —NRC(O)O—, —NRC(O)NR—,—NCO, —CHOHCHOH—, CH₂OCHCH₂O—, —(CH₂CH₂O)_(s)— and —(CH₂CH₂CH₂O)_(s)—,—C(O)—, —C(O)O—, —CH₂C(O)CH₂—, —S—, —SS—, —CHOH—, —O—, —SO—, —SO₂—,—SO₃—, —OSO₃, —SO₂NR—, —NR_(q)—, and —NR_(q) ⁺—, where R is not H, —CN,—NC, —CHOCH—, —NHC(NH)NH—, —NO₂, —NO, —OPO₃—, —OH, where s is 1-12; andR is hydrogen, C₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl, C₁₋₁₂branched, unbranched, or cyclic hydrocarbyl; P is

L is a bond or a C₁₋₁₂ branched, unbranched, or cyclic hydrocarbyl; andwherein the order of [—CH₂—CR-L-A-P] and [—CH₂—CR-L-A] is random, blockor a combination thereof and n and m are positive integers.
 2. Thepolymer modified porous substrate of claim 1, wherein the poroussubstrate is in the form of a monolith, agglomerated particles, or wovenor nonwoven fibers.
 3. The polymer modified porous substrate of claim 2,wherein the porous substrate is a glass fiber monolith.
 4. The polymermodified porous substrate of claim 1, wherein the polymer modifiedporous substrate exhibits flow rates of at least 40 mL/min through adisk having a diameter of about 47 mm.
 5. The polymer modified poroussubstrate of claim 1, wherein the polymeric monolith is functionalizedwith a polar functionality after the polymeric monolith is formed on theporous substrate.
 6. The polar functionalized polymer modified poroussubstrate of claim 5, wherein the polar functionality is selected fromthe group consisting of —NRC(O)—, —C(O)NR—, —OC(O)NR—, —OC(O)R,—NRC(O)O—, —NRC(O)NR—, —NCO, —CHOHCHOH—, CH₂OCHCH₂O—, —(CH₂CH₂O)_(s)—,—(CH₂CH₂CH₂O)_(s)—, —C(O)—, —C(O)O—, —CH₂C(O)CH₂—, —S—, —SS—, —CHOH—,—O—, —SO—, —SO₂—, —SO₃—, —OSO₃, —SO₂NH—, —SO₂NR—, —NR_(q)— and —NR_(q)⁺—, where R is not H, —CN, —NC, —CHOCH—, —NHC(NH)NH—, —NO₂, —NO, —OPO₃—,—OH; wherein s is 1-12; q is 0-3; and R is hydrogen, C₅₋₁₀ monocyclic orbicyclic aryl or heteroaryl, C₁₋₁₂ branched, unbranched, or cyclichydrocarbyl.
 7. The polar functionalized polymer modified poroussubstrate of claim 6, comprising a mixture of polar functional groups.8. The polar functionalized polymer modified porous substrate of claim7, wherein the mixture of polar functional groups is selected fromamide+amine, amide+sulfonic acid, nitrile+sulfonic acid, oramide+quaternary amine.
 9. The polar functionalized polymer modifiedporous substrate of claim 6, wherein the polar functionality is anamide, amine, nitro, quaternary amino or sulfonic acid.
 10. The polarfunctionalized polymer modified porous substrate of claim 5, wherein thepolar functionality is a cation exchange functionality or an anionexchange functionality.
 11. The polar functionalized polymer modifiedporous substrate of claim 5 associated with a support.
 12. The polarfunctionalized polymer modified porous substrate of claim 11, whereinthe support is a syringe barrel cartridge, a chromatography column, amicrofluidics platform, one or more additional membranes, a pipette tipor a multiwelled plate.
 13. The polymer modified porous substrate ofclaim 1 associated with a support.
 14. The polymer modified poroussubstrate of claim 13, wherein the support is a syringe barrelcartridge, a chromatography column, a microfluidics platform, one ormore additional membranes, a pipette tip or a multiwelled plate.
 15. Thepolymer modified porous substrate of claim 1, wherein the polymericmonolith comprises polystyrene, poly(styrene divinylbenzene), copolymerscomprising styrene or divinylbenzene and halogenated or alkylatedstyrenes, pyridines, thiophenes, furans, or imidazoles.
 16. The polymermodified porous substrate of claim 15, wherein the polymer modifiedporous substrate is polar functionalized after the polymeric monolith isformed on the porous substrate.
 17. The polar functionalized polymermodified porous substrate of claim 16, wherein the polar functionalityis selected from the group consisting of —NRC(O)—, —C(O)NR—, —OC(O)NR—,—OC(O)R, —NRC(O)O—, —NRC(O)NR—, —NCO, —CHOHCHOH—, CH₂OCHCH₂O—,—(CH₂CH₂O)_(s)—, —(CH₂CH₂CH₂O)_(s)—, —C(O)—, —C(O)O—, —CH₂C(O)CH₂—, —S—,—SS—, —CHOH—, —O—, —SO—, —SO₂—, —SO₃—, —OSO₃, —SO₂NH—, —SO₂NR—, —NR_(q)—and —NR_(q) ⁺—, where R is not H, —CN, —NC, —CHOCH—, —NHC(NH)NH—, —NO₂,—NO, —OPO₃—, —OH; wherein s is 1-12; q is 0-3; and R is hydrogen, C₅₋₁₀monocyclic or bicyclic aryl or heteroaryl, C₁₋₁₂ branched, unbranched,or cyclic hydrocarbyl.
 18. The polar functionalized polymer modifiedporous substrate of claim 16, wherein the polar functionality isselected from an amide, amine, nitro, quaternary amino or sulfonic acid.19. An amide functionalized polymer modified porous substrate preparedby the steps comprising: (a) contacting a porous substrate with asolution comprising one or more hydrophobic monomers, a porogenicsolvent and a polymerization initiator; (b) heating the porous substrateand retained solution in the absence of oxygen to polymerize the one ormore monomers onto the porous substrate to form the polymer modifiedporous substrate; (c) nitrating the polymer modified porous substrate toform the nitrated polymer modified porous substrate; (d) reducing thenitrated polymer modified porous substrate to form the aminated polymermodified porous substrate; and (e) acylating the aminated polymermodified porous substrate with an acid, an acid chloride or an acidanhydride to form the aminated polymer modified porous substrate. 20.The amide functionalized polymer modified porous substrate of claim 19associated with a support.
 21. The amide functionalized polymer modifiedporous substrate of claim 20, wherein the support is a syringe barrelcartridge, a chromatography column, a microfluidics platform, one ormore additional membranes, a pipette tip or a multiwelled plate.